Hemoglobin, 33(6):417–421, (2009)Copyright © Informa UK Ltd.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630260903327817

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LHEM0363-02691532-432XHemoglobin, Vol. 33, No. 6, Sep 2009: pp. 0–0Hemoglobin

ORIGINAL ARTICLE

A NOVEL POLYMORPHISM CAUSES A DIFFERENT RESTRICTION

PATTERN BY RsaI IN THE b-GLOBIN GENE CLUSTER: APPLICATION

IN PRENATAL DIAGNOSIS

A Novel Polymorphism in the b-Globin Gene ClusterA. Valaei et al.

Atefeh Valaei,1 Fatemeh Bayat,1 Alireza Kordafshari,1 Sirous Zeinali,1,2 and

Morteza Karimipoor1,2

1National Reference Centre For Prenatal Diagnosis of Thalassaemia and Haemoglobinopathies, Pasteur Institute of Iran, Tehran, Iran2Molecular Medicine Department, Biotechnology Research Centre, Pasteur Institute of Iran, Tehran, Iran

� b-Thalassemia (b-thal) is a major health problem in Iran and the incidence of carriers isaround 3–4%. The disease is caused by heterogeneous mutations in the b-globin gene and is char-acterized by hypochromic microcytic anemia. The human b-globin complex spans a region of 70 kband contains over 20 restriction fragment length polymorphisms (RFLPs). At least nine RFLPmarkers including RsaI/b in the b-globin gene cluster have been routinely exploited for prenataldiagnosis. Here, we report a novel polymorphism upstream of the b-globin gene characterized byRsaI digestion. Sequencing of a fragment containing this area showed a nucleotide change (T>C)at position -223 upstream of the b-globin gene. This change could interfere with precise interpreta-tion of the RsaI digestion pattern in linkage analysis and prenatal diagnosis of b-thal.

Keywords b-Globin, b-Thalassemia (b-thal), Restriction fragment length polymorphism(RFLP)

INTRODUCTION

The b-globin gene (HBB), found in the b-globin cluster, is located atthe short arm of chromosome 11 (11p15) (1). b-Thalassemia (b-thal)(OMIM:#141900) is an autosomal recessive disorder caused by more than200 different mutations in the b-globin gene, commonly prevalent in the

Received 17 June 2008; Accepted 16 April 2009.Address correspondence to Morteza Karimipoor, MD, PhD, Molecular Medicine Department,

Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran, 13164; Tel: +98-21-66480780;Fax: +98-21-66480780; E-mail: mortezakarimi@pasteur.ac.ir; mortezakarimi@yahoo.com

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tropical and subtropical regions of the world (2,3). The disease causes ane-mia with different severities which affects individuals in early childhood.These mutations are regionally specific and the spectrum of the mutationshas been characterized in most at-risk populations.

It is the most common monogenic disorder in Iran and about 3–4% ofthe people in the country are carriers of the disease, living mainly on thenorthern and southern coasts. More than 18,000 individuals affected withb-thal major and intermedia have been registered in the country (4).Thalassemia prevention programs have been in effect in the country since1997, using a premarital screening strategy (5). It is estimated that thisprogram has reduced the incidence of newborn cases by 70%. In addition,the programs have provided invaluable help for the molecular characteriza-tion of the disorder in this country. Prenatal diagnosis of b-thal usuallyrelies on mutation finding and linkage analysis using different polymorphicmarkers in the b-globin gene cluster.

The cluster contains five functional genes, 5′-e-Gg-Ag-d-b-3′, which arearranged in the order of their developmental expression from embryonicto adulthood. There are at least nine polymorphic restriction fragmentlength polymorphism (RFLP) markers distributed all over the cluster.These markers are used routinely for carrier testing, prenatal diagnosis andhaplotype analysis.

The RsaI/b polymorphism is located at 551 bp 5′ to the b-globin gene(chr 11: nt. 5205428 or nt. 61586 at accession No: U01317). The change of t(wild type) to c allele produces a restriction site for the RsaI enzyme. Thispolymorphism was searched for at dbSNP by the SNP blast program and noentry was retrieved. This RsaI site is contained within 10.3 kb or less area ofrandomization separating the 5′ and 3′ haplotype clusters (6). Working onthis RFLP marker for Iranian b-thal carriers and their families, we observeda novel polymorphism 330 bp downstream of RsaI/b, which again abolishesthe restriction site of the RsaI enzyme. Here we report the molecular char-acterization of this polymorphism.

SUBJECTS AND METHODS

This study includes b-thal carrier couples and their families referredfrom primary health care centers for mutation analysis and prenatal diagno-sis in Tehran, the capital city of Iran. Our laboratory at the Pasteur Instituteof Iran is the national reference center for DNA-based prenatal diagnosis ofthalassemia and hemoglobinopathies. All the subjects were clinically normalwith no history of jaundice, blood transfusion or other clinical complica-tions. All individuals gave written informed consent before peripheral bloodwas collected in tubes containing EDTA as anticoagulant, for DNA analysis,complete blood count (CBC) and hemoglobin (Hb) electrophoresis.

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A Novel Polymorphism in the b-Globin Gene Cluster 419

Complete blood count was performed by an automated cell counter(Sysmex Kx-21; Sysmex Corporation, Kobe, Japan), as recommended by themanufacturer. For Hb electrophoresis, erythrocyte lysates were analyzed bycellulose acetate electrophoresis (7). The lysates were also applied to col-umn chromatography to quantitate Hb A2 levels.

Genomic DNA was extracted from peripheral blood leukocytes by thesalting out method (8). Amplification refractory mutation system poly-merase chain reaction (ARMS-PCR) was exploited for detection of commonb-globin gene mutations in the Iranian population including: IVS-II-1 (G>A),IVS-I-5 (G>C), frameshift codons (FSC) 8/9 (+G) and FSC 36/37 (–T) (4).Primers were selected from Weatherall and Clegg (9). The PCR productswere run on a 2% agarose gel and bands were visualized on an UV transillu-minator after staining with ethidium bromide. When mutations were notdetected, direct sequencing of the whole b-globin gene was performed.

For genotyping of the RsaI/b RFLP marker, a 1200 bp PCR product(61504–62703 from GenBank accession No. U01317) was amplified using apublished report for primer sequences and PCR conditions (9). Digestionof the PCR product was performed by the RsaI restriction enzyme asrecommended by the manufacturer. Direct sequencing of this fragment wasperformed using the BigDye Terminator Cycle Sequencing Ready Reactionkit on an ABI PRISM™ 3130 Genetic Analyzer (Applied Biosystems, FosterCity, CA, USA) by the sense and antisense primers. The PCR-RFLP for thismarker was performed on 200 chromosomes from the normal populationand the allele frequency was calculated.

RESULTS AND DISCUSSION

The aim of this study was the molecular characterization of a differentdigestion pattern by the RsaI enzyme. In more than 400 families referred toour center for mutation analysis and prenatal diagnosis of b-thal and hemo-globinopathies in last 3 years, 10 individuals from nine families selectedwith a different digestion pattern at the RsaI/b polymorphic markers. Inour laboratory, this RFLP marker has been used extensively for linkageanalysis and prenatal diagnosis of b-thal. Of these individuals, eight werecarriers of b-thal mutations and two normal (family members of theprobands). The carriers had different mutations in the HBB gene, i.e.,c.−138 C>A (two cases), Cd30, c.89G>A (two cases), c.93 −21G>A andc.92+6T>C, c.118 C>T and c.27–28insG.

When the allele T is present at position −511 of the b-globin gene (chr11: nt. 5205428), the recognition site for the RsaI enzyme is absent anddigestion of the PCR product (1200 bp: 61504–62703 from GenBank acces-sion No. U01317) results in a 411 bp fragment and two constant fragments(95 and 694 bp) (Figure 1). In the presence of the restriction site (allele C),

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the 411 bp fragment is digested into two 81 and 330 bp fragments. In these10 individuals, an additional fragment of around 500 bp was also detected.DNA sequencing of the above PCR product in these individuals revealed aheterozygosity at nucleotide (nt) 411 of the amplified fragment (chr 11: nt5205101or nt 61914 of accession No. U01317) in all individuals. This ntchange (T>C) abolishes one of the constant restriction sites and produces a506 bp fragment (sum of 411 and 95) (Figure 1). Interestingly, all the indi-viduals with this pattern did not have a restriction site at the polymorphicsite of RsaI (T/T genotype).

Genotype analysis of this variation in 100 normal individuals showed itin three chromosomes in the heterozygous state. As mentioned, this varia-tion is not specific to a certain mutation in the HBB gene and the 10 indi-viduals had eight different mutations. Our findings suggest this nt change isnot linked to a specific mutation in the HBB gene and should be commonin the population. On the other hand, using this primer set, as suggested byWeatherall and Clegg (9), for linkage analysis and prenatal diagnosis ofb-thal and hemoglobinopathies could interfere with precise interpretationof the RsaI/b RFLP marker.

ACKNOWLEDGMENTS

The authors wish to thank the carrier couples and families for their cooper-ation and staff from the Primary Health Care clinics for referring the couples.

Declaration of Interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of this article.

FIGURE 1 Digestion pattern of the RsaI enzyme; [+] denotes the presence of the restriction site. Lane1: [−/+]; lane 2: [+/+]; lane 3: [−/−]; lane 4: [−/−] with a novel variation; lane 5: uncut fragment (1200bp); B: no template control, and M: 100 bp ladder.

1200 bp

694 bp506 bp411 bp330 bp

1 2 3 4 5 B M

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REFERENCES

1. Baysal E, Carver MFH. The b- and d-thalassemia repository (eighth edition). Hemoglobin.1995;19(3–4):213–236.

2. Patrinos GP, Giardine B, Riemer C, et al. Improvements in the HbVar database of human hemoglo-bin variants and thalassemia mutations for population and sequence variation studies. Nucleic AcidsRes. 2004;32(Database issue):D537-D541 (http://globin.cse.psu.edu).

3. Hardison RC, Chui DHK, Giardine B, et al. HbVar: A relational database of human hemoglobin vari-ants and thalassemia mutations at the globin gene server. Hum Mutat. 2002;19(3):225–233 (http://globin.cse.psu.edu).

4. Abolghasemi H, Amid A, Zeinali S, et al. Thalassemia in Iran: epidemiology, prevention and man-agement. J Pediatr Hematol Oncol. 2007;29(4):233–238.

5. Samavat A, Modell B. Iranian national thalassaemia screening programme. BMJ.2004;329(7475):1134–1137.

6. Orkin SH, Kazazian HH, Antonarakis SE, et al. Linkage of b-thalassaemia mutations and b-globingene polymorphisms with DNA polymorphisms in human b-globin gene cluster. Nature.1982;296(5858):627–631.

7. Huisman THJ, Jonxis JHP. The Hemoglobinopathies Techniques of Identification. Clinical and Biochemi-cal Analysis, Vol. 6. New York: Marcel Dekker, Inc. 1977.

8. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from humannucleated cells. Nucleic Acids Res. 1988;16(3):1215.

9. Weatherall DJ, Clegg JB. The Thalassaemia Syndromes, 4th ed. Oxford: Blackwell, 2001.

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